Reduced Activity of Anabolizing Enzymes in 5‐Fluorouracil‐resistant Human Stomach Cancer Cells

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Abstract
The mechanism of resistance to 5‐fluorouracil (5‐FU) was studied with NUGC‐3/5FU/L, a human stomach cancer cell line which had acquired resistance as a consequence of repeated 5‐day exposures to stepwise‐increasing concentrations of 5‐FU in vitro. NUGC‐3/5FU/L was 200‐fold and over 16‐fold resistant to 96‐h and 1‐h exposures to 5‐FU, respectively. NUGC‐3/5FU/L incorporated less 5‐FU into RNA, indicating resistance to the RNA‐directed action of 5‐FU. On the other hand, NUGC‐3/5FU/L also showed resistance to in situ thymidylate synthase (TS) inhibition by 5‐FU. Polymerase chain reaction‐single‐strand conformation polymorphism analysis of TS cDNA and a FdUMP ligand binding assay showed that quantitative and qualitative alterations of TS are not responsible for this resistance. In contrast, the ability to metabolize 5‐FU to its active metabolites, FUTP and FdUMP, was reduced in NUGC‐3/5FU/L. We found that not only the activities of uridine phosphorylase/kinase and orotate phosphoribosyl‐transferase (OPRT), but also the level of phosphoribosyl pyrophosphate, a cosubstrate for OPRT, were significantly lower in NUGC‐3/5FU/L than in the parent NUGC‐3. These results indicated that resistance to 5‐FU in NUGC‐3/5FU/L is due to reduced activities of 5‐FU‐anabolizing enzymes, but not to an alteration of TS. 2′‐Deoxyinosine effectively enhanced TS inhibition by 5‐FU in the resistant cells, thus markedly sensitizing them to 5‐FU.